The purpose of this R21 application is to develop a model to test the hypothesis that neutralizing antibodies re-formed during acute hepatitis C virus (HCV) infection. Comprehension of immune responses in acute HCV infection is central to efforts to treat and prevent infection. While there are several studies underway detailing the cellular response to acute infection, there is little information regarding humoral immunity. We have overcome two of the major challenges in researching acute HCV infection by establishing a repository of serum samples taken before, during and after acute infections among young injection drug users and assembling a panel of key HCV antigens to evaluate responses. Using these resources we will characterize the humoral immune response to acute HCV infection, examining the kinetics and specificities of anti-E1 and -E2 glycoprotein responses, as well as IgG class switching. These events will be correlated with liver enzyme changes, HCV RNA titer, and HCV sequence evolution. We have established 5.2kb clones representing the major and minor viral variants and have sequenced the major variants at the onset of viremia and 6 months thereafter for 18 individuals. These reagents and the corresponding serum repository provide the rare opportunity to examine strain specific responses. Therefore, a second aim of this investigation is to construct a pseudotype virus that readily expresses HCV envelope sequences using lentiviral replicative machinery, as has been done recently with other viruses. The hypothesis that antibody to envelope reduces the fitness of individual HCV variants (neutralization) will be examined by expressing the envelope sequences of major and minor variants of 4 or more persons at two time points, beginning with the onset of viremia and 6 months later. In addition, equal numbers of 1a and 1b infected participants will be used for analysis. We will analyze pseudotyped virus by correlating envelope sequence with the ability of serum collected at various times to neutralize its infectivity. We believe there is a high likelihood this research will be successful, but submit the work initially as an R21 to prove the feasibility of the pseudotype system.